Mechanisms of high-level fosfomycin resistance in Staphylococcus aureus epidemic lineage ST5

Abstract

Objectives: Fosfomycin resistance has become a clinical concern. In this study, we analysed the dynamic change of fosfomycin MIC in the epidemic Staphylococcus aureus lineages in a teaching hospital in Shanghai for 12 years and sought to elucidate the major underlying mechanisms.

Methods: MLST was conducted for 4580 S. aureus isolates recovered from 2008 to 2019. Fosfomycin MIC was determined by the agar dilution method. The genome data of 230 S. aureus epidemic lineage isolates were acquired from a next-generation sequencing (NGS) platform. Gene deletion and corresponding complementation mutants were constructed to confirm the mechanism of fosfomycin resistance.

Results: The predominant S. aureus lineages during the past 12 years were ST5 and ST239 (45.6%; 2090/4580). However, ST5 has been spreading clinically, while ST239 has gradually disappeared recently. Consistent with epidemic trends, fosfomycin-resistant ST5 increased from 19.5% to 67.3%. Most fosfomycin-resistant ST5 isolates (92.7%; 647/698) possessed high-level resistance (MIC > 1024 mg/L) with combined mutations mainly in glpT and uhpT. In contrast, fosfomycin-resistant ST239 isolates (76.8%; 149/194) mainly acquired low-level resistance (MIC = 64-128 mg/L) with mutation primarily in hptA. Deletion of a single resistant gene merely resulted in low-level fosfomycin resistance, while double-gene mutants ΔglpTΔuhpT, ΔglpTΔhptA and ΔglpTΔhptR acquired high-level fosfomycin resistance.

Conclusions: The high-level fosfomycin resistance of S. aureus epidemic lineage ST5 is mainly due to the accumulation of mutations in the resistant genes related to membrane transporter systems, and partly contributes to its persistent prevalence under clinical antibiotic pressure.

Figure 1.

The epidemiology characteristics of S. aureus lineages, from 2008 to 2019. (a) The dynamic changes of lineages of S. aureus recovered from the study period. Blocks are coloured to reflect the proportion of corresponding lineages. (b) Isolation rates of two epidemic S. aureus lineages, ST5 and ST239. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 2.

Resistance rates of commonly used antibiotics for S. aureus ST5 (red circles) and ST239 (black squares). The chosen antibiotics included penicillin, cefazolin, cefoxitin, erythromycin, fosfomycin, gentamicin, levofloxacin, linezolid and vancomycin. All isolates were susceptible to linezolid and vancomycin (not shown here). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 3.

Comparison of fosfomycin MICs between ST5 and ST239 fosfomycin-resistant S. aureus isolates. Six different MIC levels are included. The number in each grid represents the proportion of different MIC levels in ST5 and ST239 during the study period. The numbers of cases of fosfomycin-resistant ST239 S. aureus isolates after 2010 were <5 (0 in 2019) and are stated altogether. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 4.

Major mutations in ST5 and ST239 fosfomycin-resistant S. aureus isolates. (a) Mutation rates of fosfomycin resistance genes in ST5 and ST239 isolates. Most mutations appeared in glpT, hptA, hptS, hptR and uhpT. (b) Major mutation types and positions in ST5 and ST239 isolates. A total of 14 mutation types were discovered in uhpT of ST5 but only 8 major mutation types are presented in the figure. The lengths of five genes are shown in the middle. DEL, deletion; INS, insertion. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 5.

Phylogenetic analysis based on MLSA and the de-duplicated tree. (a) Phylogenetic tree of 168 S. aureus ST5 isolates, including both susceptible (≤32 mg/L, the green part of the circular multi-coloured strip) and resistant phenotypes, was built with IQ-TREE. The circular multi-coloured strip reflects the MIC levels (shown in the MIC legend). The blue bars outside the strip indicate the number of the mutated genes in every isolate (from 0 to 3). Two clades with low fosfomycin resistance are characterized by clade I and clade II. (b) The de-duplicated phylogenetic tree of 21 distinct genotypes. The multi-coloured strip reflects the MIC levels (shown in the MIC legend). The heatmap reveals the genes mutated in the genotypes (WT in white and mutant in black). The blue bars on the far right indicate the number of each genotype. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.